【摘要】 目的获得高纯度的Vif-ΔN28和Cullin5-N138蛋白。方法以Vif/VR1012质粒为模板,PCR扩增出Vif-ΔN28基因并插入pRSETB质粒。将构建的原核表达载体Vif-ΔN28/pRSETB以及已有的Cullin5-N138/pRSETB质粒分别转化大肠杆菌BL21(DE3),经IPTG诱导表达,产物经Ni-NTA离子纯化柱纯化、复性。结果所表达的Vif-ΔN28和Cullin5-N138蛋白约占各自菌体总蛋白的20%,纯化后蛋白纯度均可达90%以上。结论已成功构建了Vif-ΔN28原核表达质粒,并在大肠杆菌中高效表达,获得了可溶性的纯化的Vif-ΔN28和Cullin5-N138蛋白。更多还原

【Abstract】 Objective To obtain highly purified Vif-ΔN28 and Cullin5-N138 proteins.Methods Amplify Vif-ΔN28 gene by PCR using plasmid Vif/VR1012 as template and insert into plasmid pRSETB.Transform the constructed recombinant plasmid Vif-ΔN28/pRSETB,as well as recombinant plasmid Cullin5-N138/pRSETB constructed in advance,to E.coli BL21(DE3) for expression under induction of IPTG.The expressed product was purified by Ni-NTA ion exchange chromatography and re-naturalized.Results Either the expressed Vif-ΔN28 or Cullin5-N138 contained 20% of total somatic protein and reached a purity of more than 90% after purification.Conclusion Recombinant plasmid Vif-ΔN28/pRSETB was successfully constructed and highly expressed in E.coli.Both purified Vif-ΔN28 and Cullin5-N138 proteins in soluble forms were obtained.更多还原